Fig 4.

mag-1 induces the accumulation of HIF-1α protein under hypoxia. (A) H1299 cells stably transfected with mag-1 or vector were cultivated under normoxia or hypoxia (0.5% O₂) for 12 hrs. HIF-1α protein was detected using Western blot. (B) Endogenous mag-1 was knocked down by specific shRNA in EMT6 cells. EMT6/mag-1 KD and scramble control cells were subjected to hypoxia for 12 hrs. Protein accumulation of HIF-1α was analysed using Western blot (upper two lines), whereas the corresponding mRNA levels of HIF-1α were assessed using RT-PCR (lower two lines). (C) H1299/mag-1 and vector control cells were subjected to hypoxia for the indicated time. The induction of mag-1 expression was analysed using Western blot. (D) Effects of mag-1 overexpression on HIF-1α half-life. mag-1-stably transfected H1299 and mock control cells were incubated in hypoxia for 8 hrs and then treated with 25 g/ml cycloheximide, after which the decay of HIF-1α was monitored at the indicated times using Western blot.